Modelling amperometric enzyme electrode with substrate cyclic conversion

A mathematical model of amperometric enzyme electrodes in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model is based on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetic of the enzymatic reaction. The digital simulation was carried out using the finite difference technique. The influence of the substrate concentration, the maximal enzymatic rate as well as the membrane thickness on the biosensor response was investigated. The numerical experiments demonstrate significant (up to dozens of times) gain in biosensor sensitivity at low concentrations of substrate when the biosensor response is under diffusion control.     

Electrochemical reduction of cytochromes P450 using electrodes containing immobilized substrates

A new approach for the electrochemical reduction of cytochromes P450 (P450, CYPs) with electrodes chemically modified with CYP appropriate substrates (“reverse” electrodes) has been proposed. The method is based on the analysis of cyclic voltammograms, square wave voltammograms, amperograms and determination of such electrochemical characteristics as catalytic current and redox potential. The differences of maximal current and potentials in square wave voltammograms and catalytic current in amperometric measurements are more sensitive and reliable. The planar mode of screen-printed electrodes permits to use 20–60 μl of electrolyte volume. We have investigated P450 2B4-benzphetamine or P450scc-cholesterol enzyme-substrate pairs. Electrochemical parameters of electrodes with nonspecific P450 substrate were differed from the electrodes with appropriate substrates.     

Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode

A new amperometric biosensor, based on adsorption of glucose oxidase (GOD) at the platinum nanoparticle-modified carbon nanotube (CNT) electrode, is presented in this article. CNTs were grown directly on the graphite substrate. The resulting GOD/Pt/CNT electrode was covered by a thin layer of Nafion to avoid the loss of GOD in determination and to improve the anti-interferent ability. The morphologies and electrochemical performance of the CNT, Pt/CNT, and Nafion/GOD/Pt/CNT electrodes have been investigated by scanning electron microscopy, cyclic voltammetry, and amperometric methods. The excellent electrocatalytic activity and special three-dimensional structure of the enzyme electrode result in good characteristics such as a large determination range (0.1-13.5mM), a short response time (within 5s), a large current density (1.176 mA cm(-2)), and high sensitivity (91mA M(-1)cm(-2)) and stability (73.5% remains after 22 days). In addition, effects of pH value, applied potential, electrode construction, and electroactive interferents on the amperometric response of the sensor were investigated and discussed. The reproducibility and applicability to whole blood analysis of the enzyme electrode were also evaluated.     

Composite electrochemical biosensors: a comparison of three different electrode matrices for the construction of amperometric tyrosinase biosensors

A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphite-Teflon; reticulated vitreous carbon (RVC)-epoxy resin; and graphite-ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite-EPD electrodes, whereas a longer useful lifetime was observed for the graphite-Teflon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphite-EPD>graphite-Teflon>RVC-epoxy resin. A correlation between sensitivity and the catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix influences the interactions in the tyrosinase catalytic cycle.     

Insulin-dependent and insulin-independent low Km cyclic AMP phosphodiesterase from rat adipose tissue

Chromatographic analysis of a soluble extract of rat adipose tissue on DEAE-Sephacel resolves four distinct peaks of 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity. Kinetic investigation indicates that two of these fractions have a high affinity for cyclic AMP and show negative cooperative kinetic behavior at high substrate concentration. They differ in the degree of inhibition by cyclic GMP and in their response to insulin. If rat epididymal fat pads are incubated with insulin prior to homogenization, only one of the low Km cyclic AMP phosphodiesterase forms is stimulated.     

Dependence of the response of an amperometric biosensor formed in a micro flow channel on structural and conditional parameters

Comprehensive analysis of the behavior of an amperometric biosensor incorporated in a micro flow channel was conducted by changing the structural and conditional parameters. The device used in the characterization consisted of a thin-film three-electrode system and a silicone rubber flow channel. An enzyme, glucose oxidase, was immobilized either at the bottom of the silicone rubber flow channel or on the electrode substrate. The flow rate, concentration, position of the immobilized enzyme, and channel height were changed, and the changes in the output current and the conversion efficiency were examined. When the flow rate and/or the channel height decreased, the output current and the conversion efficiency significantly increased. The conversion efficiency also increased by decreasing the concentration. The tendency of the flow dependence was reversed when the position of the immobilized enzyme was changed from the silicone rubber side to the electrode substrate. In addition, the influence of l-ascorbic acid was reduced by placing additional working electrodes in the upper stream. l-Ascorbic acid was eliminated more effectively as the flow rate decreased and the area of the working electrode for elimination increased.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 μl each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Gradient liquid chromatography of leucine-enkephalin peptide and its metabolites with electrochemical detection using highly boron-doped diamond electrode

Boron-doped diamond thin film (BDD) electrodes have been used to study the oxidation reactions and to detect leucine-enkephalinamide (LEA) and its metabolites, tyrosine (T), tyrosyl-alanine (TA), tyrosyl-alanine-glycine (TAG) and leucine-enkephalin (LE) using cyclic voltammetry (CV), flow-injection analysis (FIA), and gradient liquid chromatography (LC) with amperometric detection. At diamond electrodes, well-defined and highly reproducible cyclic voltammograms were obtained with signal-to-background (S/B) ratios 5-10 times higher than those observed for glassy carbon (GC) electrodes. The analytical peaks of LC for LEA and its metabolites were well resolved. No deactivation of BDD electrodes was found after several experiments with standard as well as plasma samples, indicating high stability of the electrode. Calibration curves were linear over a wide range from 0.06 to 30 microM with regression coefficients of 0.999 for all compounds. The limits of detection obtained based on a signal-to-noise ratio of 3:1 were 3, 2.2, 2.7, 20 and 11 nM for T, TA, TAG, LE and LEA, respectively. These values were at least one order lower than those obtained at GC electrodes, which has given limits of detection of 22.88, 20.64, 89.57, 116.04 and 75.67 for T, TA, TAG, LE and LEA, respectively. Application of this method to real samples was demonstrated and validated using rabbit serum samples. This work shows the promising use of conducting diamond as an amperometric detector in gradient LC, especially for the analysis of enkephalinamide and its metabolites.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 &#119 l each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Kinetic model for dynamic response of three-phase fluidized bed biofilm reactor for wastewater treatment

Step changes in inlet concentration has been introduced into the completely mixed three-phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the dynamic behavior of the system and to establish the suitable kinetic model from the response curve. Three identical reactors having different biomass volumes were operated in parallel. It was found that the response curves showed second-order characteristics, and thus at least two first-order differential equations are necessary to simulate the substrate and biomass response curves. Nonlinear regression analysis was performed using different types of rate equations and their corresponding kinetic parameters were used to simulate the theoretical response curve using the Runge–Kutta numerical integration method. As a result, although various types of conventional biokinetic models such as Monod, Haldane and Andrew types were examined, all the theoretical substrate response curves underestimated time constants compared to the actual substrate response plots. On the other hand, the theoretical curve of the kinetic model that incorporates adsorption term has best fit to the actual response in most of the cases. Thus, it was concluded that adsorption of substrate onto biofilm and carrier particles has significant effect on the dynamic response in biofilm processes.     

Studies on the electrochemical performance of glucose biosensor based on ferrocene encapsulated ORMOSIL and glucose oxidase modified graphite paste electrode

The electrochemical performance of a new glucose biosensor is reported. The glucose biosensor is developed using glucose oxidase (GOD) and ferrocene encapsulated palladium (Pd)-linked organically modified sol-gel glass (ORMOSIL) material incorporated within graphite paste electrode. The ORMOSIL material incorporated within graphite paste electrode behaves as an excellent electrocatalyst for the oxidation of enzymatically reduced GOD. The electrochemical behavior of new glucose biosensor has been examined by cyclic volammetry and amperometric measurements. The bioelectrocatalysis of ORMOSIL embedded within graphite paste as a function of storage time and varying concentration of ORMOSIL is reported. The initial amperometric response on glucose sensing is recorded to be 145 microA at 15% (w/w) concentration of the ORMOSIL which is decreased to 20 microA at 5% of the same keeping GOD concentration constant. The variation of electrochemical behavior of the ORMOSIL embedded within graphite paste as a function of time has also been studied based on cyclic voltammetry. The voltammograms showing the reversible electrochemistry of ORMOSIL encapsulated ferrocene is changed into a plateau shape as a function of time, however, the electrocatalytic behavior is still retained. The practical usability of new glucose sensor has been compared with earlier developed glucose sensor. The sensitivity, response time and linearity of the new glucose biosensor are found to be excellent over earlier reported glucose biosensor. The amperometric response, calibration curve and practical applications of new glucose sensor are reported.     

Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel

A novel amperometric biosensor, based on electrodeposition of platinum nanoparticles onto multi-walled carbon nanotube (MWNTs) and immobilizing enzyme with chitosan-SiO(2) sol-gel, is presented in this article. MWNTs were cast on the glass carbon (GC) substrate directly. An extra Nafion coating was used to eliminate common interferents such as acetaminophen and ascorbic acids. The morphologies and electrochemical performance of the modified electrodes have been investigated by scanning electron microscopy (SEM) and amperometric methods, respectively. The synergistic action of Pt and MWNTs and the biocompatibility of chitosan-SiO(2) sol-gel made the biosensor have excellent electrocatalytic activity and high stability. The resulting biosensor exhibits good response performance to glucose with a wide linear range from 1 microM to 23 mM and a low detection limit 1 microM. The biosensor also shows a short response time (within 5s), and a high sensitivity (58.9 microAm M(-1)cm(-2)). In addition, effects of pH value, applied potential, rotating rate, electrode construction and electroactive interferents on the amperometric response of the sensor were investigated and discussed in detail.     

D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity

For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.     

A mediatorless and label-free amperometric immunosensor for detection of h-IgG

A novel experimental methodology for studying a mediatorless and label-free immunosensor is proposed by immobilizing antibody on gold nanoparticle/L-cysteine coated electrode (nano-Au/L-cysteine electrode). Differential pulse voltammograms (DPV) resulting from the assembled immunosensor indicate that the immunosensor shows excellent electrochemical response to dopamine so that the electrochemical response is utilized for the signal generation step of the immunosensor. Therefore, by means of unenzymatic-labeling procedure combined with the amperometric detection using dopamine as substrate, the immunological reaction can be detected. After the immunosensor is incubated with h-IgG solution, the access of electrocatalytic behavior center of the immunosensor to dopamine is partly inhibited, which leads to a linear decrease in amperometric response of the immunosensor with h-IgG concentration over a range 0.82-90 ng mL(-1) by DPV.     

Amperometric responses to acetylene, ethylene, and methane by a Clark-type oxygen electrode

Hydrogen-conditioned Clark electrodes exposed to successive acetylene samples displayed a decreasing curvilinear response. Slight electrode responses are also effected by saturated ethylene solutions. Thus, amperometric measurements of hydrogen or carbon monoxide in the aqueous environment by these electrodes are perturbated by the presence of acetylene and to a much lesser extent by ethylene. Methane-saturated solutions induced no electrode response.     

Combined effects of microtopography and cyclic strain on vascular smooth muscle cell orientation

Cellular alignment studies have shown that cell orientation has a large effect on the expression and behavior of cells. Cyclic strain and substrate microtopography have each been shown to regulate cellular alignment. This study examined the combined effects of these two stimuli on the alignment of bovine vascular smooth muscle cells (VSMCs). Cells were cultured on substrates with microgrooves of varying widths oriented either parallel or perpendicular to the direction of an applied cyclic tensile strain. We found that microgrooves oriented parallel to the direction of the applied strain limited the orientation response of VSMCs to the mechanical stimulus, while grooves perpendicular to the applied strain enhanced cellular alignment. Further, the extent to which parallel grooves limited cell alignment was found to be dependent on the groove width. It was found that for both a small (15microm) and a large (70microm) groove width, cells were better able to reorient in response to the applied strain than for an intermediate groove width (40microm). This study indicates that microtopographical cues modulate the orientation response of VSMCs to cyclic strain. The results suggest that there is a range of microgroove dimensions that is most effective at maintaining the orientation of the cells in the presence of an opposing stimulus induced by cyclic strain.     

Electrochemistry of immobilized CuZnSOD and FeSOD and their interaction with superoxide radicals

Copper, zinc superoxide dismutase (CuZnSOD) from bovine erythrocytes and iron superoxide dismutase from Escherichia coli (FeSOD) were immobilized on 3-mercaptopropionic acid (MPA)-modified gold electrodes, respectively. The characterization of the SOD electrodes showed a quasi-reversible, electrochemical redox behavior with a formal potential of 47+/-4 mV and -154+/-5 mV (vs. Ag/AgCl, 1 M KCl) for surface adsorbed CuZnSOD and FeSOD, respectively. The heterogeneous electron transfer rate constants were determined to be about 65 and 35/s, respectively. Covalent fixation of both SODs was also feasible with only slight changes in the formal potential. The interaction of superoxide radicals (O(2)(-)) with the SOD electrode was investigated. No catalytic current could be observed. However, due to the fast cyclic redox reaction of SOD with superoxide, the communication of the protein with the electrode was strongly influenced. The amperometric detection of superoxide radicals is discussed.     

A new format of electrodes for the electrochemical reduction of cytochromes P450

New approach to the electrochemical reduction of cytochromes P450 (P450s, CYPs) at electrodes chemically modified with appropriate substrates for P450s ("reverse" electrodes) was proposed. The method is based on the analysis of cyclic voltammograms, square-wave voltammograms and amperograms with subsequent determination of electrochemical characteristics such as catalytic current and redox potential. The sensitivity of proposed method is 0.2-1 nmol P450/electrode. The changes of maximal current and of redox potentials in square-wave voltammograms as well as the changes of catalytic current in amperometric experiments proved to be informative and reliable. Planar regime of screen-printed electrodes (strip-type sensors) enabled to utilise 20-60 microl of electrolyte volume. The enzyme-substrate pairs P450 2B4/benzphetamine and P450scc/cholesterol were investigated. Electrochemical parameters of electrodes with unspecific P450 substrates differed considerably from electrodes with appropriate substrates.     

Application of electrochemically prepared polypyrrole-polyvinyl sulphonate films to DNA biosensor

Double stranded calf thymus deoxyribonucleic acid (DNA) was physisorbed onto polypyrrole-polyvinyl sulphonate (PPY-PVS) films electrochemically deposited onto indium-tin-oxide (ITO) coated glass plates. These DNA immobilized PPY-PVS films optimized for various conditions, such as polymerization potential, pH of buffer, DNA concentration and scan rate were characterized using Fourier-transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM) and cyclic voltammetry (CV) techniques, respectively. The amperometric response studies of these DNA/PPY-PVS electrodes were carried out as a function of 2-aminoantharcene (2-AA, 0.01-20 ppm) and o-chlorophenol (OCP, 0.1-30 ppm) concentration, respectively at 25 degrees C. The observed amperometric current arising due to oxidation of guanine in the DNA/PPY-PVS films decreased linearly with the increase in the concentration of 2-AA and OCP. It has been revealed that 10 ppm of 2-AA is sufficient to reduce the observed guanine oxidation peak current by approximately -95+/-10% as compared to the reported values. A 25 ppm of OCP was capable enough to reduce the guanine oxidation current to zero. These DNA/PPY-PVS electrodes were found to have a shelf life of about 4 months when stored at 25 degrees C.